ABSTRACT Protein synthesis (translation) constitutes one of the most fundamental of all cellular processes. The balance between protein synthesis and protein degradation is critical in maintaining cellular homeostasis. Translation is one of the most energy consumptive processes in a cell, accounting for as much as 30% of the energy usage in a eukaryotic cell, making its tight regulation a necessity. Eukaryotic elongation factor 2 kinase (eEF-2K), a unique member of the ?-kinase family, is a key regulator of the elongation phase of translation. eEF-2K phosphorylates and inactivates elongation factor 2 (eEF-2), leading to a reduction in global translation rates on one hand and differential translation of certain proteins on the other. The activity of eEF-2K is dependent on calmodulin, and is subject to complex regulation by calcium ions and phosphorylation . While there is accumulating evidence that the dysregulation of eEF-2K activity is involved in several disease states (e.g. Alzheimer's disease, depression, and a variety of cancers), a detailed mechanistic understanding of eEF-2K activation and regulation is lacking. Our long-term goal is to precisely define the mechanisms by which eEF-2K is activated and regulated; such information is critical to understanding its contributions to both normal cellular processes and in the etiology and progression of disease states. Toward this goal, we recently identified calmodulin-stimulated autophosphorylation on a specific residue, T348, as being the key step in the activation of eEF-2K. Our objective in the present proposal is to describe the structural and biochemical mechanisms of eEF-2K activation by calmodulin and the roles of calcium and phosphorylation at a regulatory site, S500, in modulating this process. We will achieve this goal through a multi-faceted and collaborative approach that encompasses enzymological, kinetic, solution-state nuclear magnetic resonance and cell-biological techniques. Our central hypothesis is that T348 autophosphorylation and activation of eEF-2K is dependent on calmodulin binding. Calcium ions and S500 phosphorylation play overlapping roles but utilize distinct mechanisms in regulating the calmodulin sensitivity of eEF-2K and thereby its activation. We expect our analyses will provide the basis for a thorough mechanistic understanding of how eEF-2K integrates these two disparate signals that regulate its activity. Such a detailed picture of eEF-2K regulation is critical for elucidating its contribution to a variety of fundamental cellular process and its deregulation in a variety of disease states.